The binding of substrates and inhibitors to wild-typeTyrosine phenol-lyase (TPL) 1 (EC 4.1.99.2) and tryptophan indole-lyase (Trpase, EC 4.1.99.1) are pyridoxal 5Ј-phosphate (PLP)-dependent enzymes that catalyze -elimination reactions to form phenol or indole and ammonium pyruvate from tyrosine and tryptophan, respectively (Schemes 1 and 2) (1, 2). The amino acid sequence alignment and crystallographic structures show that these two enzymes are very closely related (1, 2). Mechanistic studies (3-5) demonstrated that TPL and Trpase follow very similar catalytic mechanisms (Scheme 3). Both enzymes can catalyze the elimination reactions in vitro of a wide range of amino acids with suitable leaving groups on the (8), and Oacyl-L-serines (7). However, in vivo, the enzymes are extremely specific for their respective physiological substrates. Both TPL and Trpase react with substrates and inhibitors to form equilibrating mixtures of external aldimine and quinonoid complexes (5, 10 -14). We have previously reported the x-ray crystallographic structure of TPL complexed with the substrate analog 3-(4Ј-hydroxyphenyl)propionic acid (15). This complex resembles the Michaelis-Menten complex, because the analog lacks an amino group and is unable to form an external aldimine. We would like to obtain structures of quinonoid intermediates for both enzymes, because quinonoid intermediates are proposed to play a central role in the mechanisms of both enzymes (Scheme 3). In previous studies with Trpase and tryptophan synthase (16, 17), we prepared inhibitors oxindolyl-Lalanine (Scheme 4, I) and dihydro-L-tryptophan, which resemble the indolenine intermediate (Scheme 3), a proposed intermediate in the reactions of both enzymes. Trpase and tryptophan synthase are inhibited by different diastereomers of dihydro-L-tryptophan, suggesting that the indolenine intermediates in the reactions of the two enzymes exhibit opposite chirality (17). Oxindolyl-L-alanine was found previously to inhibit Escherichia coli Trpase with a K i value of ϳ2.5-6 M (3, 16, 18) and to form a prominent absorption band at 502 nm, demonstrating the predominant formation of a stable quinon-